Anisotropic NMR Research Articles

Artifact-free measurement of residual dipolar couplings in DMSO by the use of cross-linked perdeuterated poly(acrylonitrile) as alignment medium

Perdeuterated poly(acrylonitrile) is introduced as a practically proton-free alignment medium for the measurement of anisotropic NMR parameters; its use in conventional glass tubes and in a Kalrez® 8002 UP-based stretching device with resulting spectra of astonishing quality are demonstrated.

New Class of Hydroxy-Substituted Squaraine Rotaxane.

A templated macrocyclization reaction was used to permanently encapsulate a highly fluorescent hydroxy-substituted squaraine dye inside a tetralactam macrocycle. The free squaraine dye is quite rigid due to internal hydrogen bonding and its photophysical properties hardly change upon encapsulation. A combination of X-ray and NMR data show that the surrounding tetralactam macrocycle adopts an unusually rigid chair conformation and does not undergo rapid pirouetting. Because of its large size and conformational rigidity, the macrocycle creates anisotropic NMR shielding zones that extend over the N,N-dibutylamino groups at each end of the squaraine thread. This shielding anisotropy allows hindered aryl-N rotation to be observed by NMR and provides direct experimental evidence that quinoid-like resonance structures are major contributors to the bis(N,N-dialkylaminophenyl)squaraine resonance hybrid.

Covalently Cross‐linked Gelatin Allows Chiral Distinction at Elevated Temperatures and in DMSO

Stretched gelatin has been known as a chiral discriminator in aqueous solution for several years, as measured by differences in anisotropic NMR parameters like residual dipolar couplings. The extension of the method to higher temperatures and the important solvent DMSO by cross-linking gelatin with accelerated electrons potentially opens the field to a variety of polar chiral molecules (see figure).

Implementation of High Resolution 43Ca Solid State NMR Spectroscopy: Toward the Elucidation of Calcium Sites in Biological Materials

Calcium is one of the most abundant cations in living organisms. It is found in the mineral phase of bone and in proteins like calmodulin. However, its exact environment beyond the first coordination sphere is often unknown, thus hampering the understanding of many biological processes. Here, calcium benzoate trihydrate (Ca(C(6)H(5)COO)(2) x 3 H(2)O) was used as a model for the NMR analysis of calcium sites in biological materials, because of the similarity of its calcium coordination, to water and carboxylate ligands, to that in several calcium-proteins. First, calcium-43 magic angle spinning (MAS) and static NMR spectra of a (43)Ca enriched sample were recorded at different magnetic fields, to investigate the electronic environment of calcium. Complex static lineshapes were obtained because of the presence of anisotropic NMR interactions of similar magnitude (chemical shift anisotropy and quadrupolar interaction), and the full interpretation of the spectra required simulations and gauge-including projector augmented wave (GIPAW) DFT calculations. An NMR investigation of the coordination environment of Ca(2+) was carried out, using high resolution (13)C-(43)Ca MAS NMR experiments such as TRAPDOR (transfer of population double resonance) and heteronuclear J-spin-echoes. It was shown that despite the weakness of (13)C-(43)Ca interactions, it is possible to discriminate carbon atoms according to their calcium environment. Long-range calcium-carbon correlations were even evidenced by TRAPDOR, reaching distances >5.6 A. This work demonstrates that by combining solid state NMR experiments, DFT calculations, and simulations, it will be possible to elucidate the electronic and coordination environment of calcium in many important and complex materials.

Tilt and Azimuthal Angles of a Transmembrane Peptide: A Comparison between Molecular Dynamics Calculations and Solid-State NMR Data of Sarcolipin in Lipid Membranes

We report molecular dynamics simulations in the explicit membrane environment of a small membrane-embedded protein, sarcolipin, which regulates the sarcoplasmic reticulum Ca-ATPase activity in both cardiac and skeletal muscle. In its monomeric form, we found that sarcolipin adopts a helical conformation, with a computed average tilt angle of 28 ± 6° and azymuthal angles of 66 ± 22°, in reasonable accord with angles determined experimentally (23 ± 2° and 50 ± 4°, respectively) using solid-state NMR with separated-local-field experiments. The effects of time and spatial averaging on both 15N chemical shift anisotropy and 1H/ 15N dipolar couplings have been analyzed using short-time averages of fast amide out-of-plane motions and following principal component dynamic trajectories. We found that it is possible to reproduce the regular oscillatory patterns observed for the anisotropic NMR parameters (i.e., PISA wheels) employing average amide vectors. This work highlights the role of molecular dynamics simulations as a tool for the analysis and interpretation of solid-state NMR data.

Effect of phase inversion of radiofrequency pulses on the recoupling of anisotropic NMR interactions under magic-angle spinning

The effect of phase inversion of radiofrequency pulses on the recoupling of anisotropic chemical shift and heteronuclear magnetic dipole–dipole interactions has been investigated by a simple analytical treatment. For the recoupling of chemical shift anisotropy, the evolution of the spin system will be different if the sign of all rf phases is inverted. This phenomenon is readily explained by the higher-order effect of average Hamiltonian theory. On the contrary, such dramatic phase inversion effect is not observed for heteronuclear dipolar recoupling, which can also be rationalized in our treatment. Numerical simulations are carried out to corroborate our analytical results and experimental verification is obtained for [1- 13C]-alanine.

Magnetically Aligned Bicelles To Study the Orientation of Lipophilic Ligands in Membrane Bilayers

Magnetically aligned bicelles were used as a model membrane to study the orientation and dynamic properties of two cannabinoids (Delta (8)-THC and Me-Delta (8)-THC) using (31)P and (2)H NMR. The uniform alignment of the bicelles allowed us to obtain well resolved deuterium spectra from a solution NMR spectrometer. The preferred orientations of Delta (8)-THC and Me-Delta (8)-THC were calculated on the basis of the measurements of individual quadrupolar splittings. Our results agree with previous experiments using multilamellar membranes as well as with molecular dynamics simulation data described here. In conjunction with our earlier report using small and fast tumbling bicelles, the present work of well aligned bicelles shows that bicelle preparations can provide either pseudoisotropic or anisotropic NMR spectra to study the conformation, orientation, and dynamic properties of ligands in membrane bilayers. Such data are of critical value for understanding the interactions of lipophilic drug molecules with membrane proteins.

Pore Structure, Thinning Effect, and Lateral Diffusive Dynamics of Oriented Lipid Membranes Interacting with Antimicrobial Peptide Protegrin-1: 31P and 2H Solid-State NMR Study

Membrane pores that are induced in oriented membranes by an antimicrobial peptide (AMP), protegrin-1 (PG-1), are investigated by (31)P and (2)H solid state NMR spectroscopy. We incorporated a well-studied peptide, protegrin-1 (PG-1), a beta-sheet AMP, to investigate AMP-induced dynamic supramolecular lipid assemblies at different peptide concentrations and membrane compositions. Anisotropic NMR line shapes specifying toroidal pores and thinned membranes, which are formed in membrane bilayers by the binding of AMPs, have been analyzed for the first time. Theoretical NMR line shapes of lipids distributed on the surface of toroidal pores and thinned membranes reproduce reasonably well the line shape characteristics of our experimentally measured (31)P and (2)H solid-state NMR spectra of oriented lipids binding with PG-1. The lateral diffusions of lipids are also analyzed from the motionally averaged one- and two-dimensional (31)P and (2)H solid-state NMR spectra of oriented lipids that are binding with AMPs.

Stereoselective synthesis of 2,3,7‐trimethylcyclooctanone and related compounds and determination of their relative and absolute configurations by the MαNP acid method

The copper/chiral phosphoramidite (L(1))-catalyzed conjugate addition of dimethylzinc to cycloocta-2,7-dienone 4, followed by the methylation of the intermediate enolate, yielded a single isomer of 7,8-dimethylcyclooct-2-enone (+)-5. Compound (+)-5 was subjected to the second conjugate addition with ent-L(1) giving only one stereoisomer of 2,3,7-trimethylcyclooctanone (+)-6, which was converted to 2,3,7-trimethylcyclooctanol 7. To determine the relative and absolute configurations of these compounds, the (1)H NMR anisotropy method using (S)-(+)-2-methoxy-2-(1-naphthyl)propionic acid {(S)-(+)-MalphaNP acid} 1 was applied. Racemic alcohol (+/-)-7 was esterified with (S)-(+)-MalphaNP acid 1 yielding diastereomeric esters, which were efficiently separated by HPLC on silica gel affording the first-eluted MalphaNP ester (-)-10a and the second-eluted one (-)-10b. The relative and absolute configurations of ester (-)-10a were determined to be (S;1R,2S,3R,7S) by analyzing the (1)H and (13)C NMR spectra of (-)-10a and (-)-10b, especially their HSQC-TOCSY and NOESY spectra, and by applying the MalphaNP anisotropy method. The alcohol 7 formed from (+)-6 was similarly esterified with (S)-(+)-MalphaNP acid 1 yielding an MalphaNP ester, which was identical with (-)-10a, and the relative and absolute configurations of 2,3,7-trimethylcyclooctanone (+)-6 were determined to be (2S,3R,7S).

Synthesis of Enantiopure Aliphatic Acetylene Alcohols and Determination of Their Absolute Configurations by 1H NMR Anisotropy and/or X‐ray Crystallography

AbstractThe MαNP acid method has been applied to racemic aliphatic acetylene alcohols in order to simultaneously prepare enantiopure alcohols and to determine their absolute configurations by 1H NMR anisotropy. Racemic acetylene alcohols 6–8, 11, and 20 were esterified with MαNP acid (S)‐(+)‐1 to yield diastereomeric MαNP esters which were efficiently separated by HPLC on silica gel with separation factors α in the range 1.60–1.93. The 1H NMR anisotropy factors Δδ [= δ(2nd fr.) – δ(1st fr.)] were calculated from the data of the first‐ (22a–27a) and second‐eluted MαNP esters (22b–27b). The absolute configurations of the first‐eluted esters were determined from the distribution of Δδ values in the MαNP sector rule. In the case of MαNP ester 26b, the assigned absolute configuration was confirmed by X‐ray crystallography. The solvolysis of MαNP esters yielded enantiopure acetylene alcohols 5–8 with established absolute configurations. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Restricted Segmental Mobility Can Facilitate Medium-Range Chain Diffusion: A NMR Study of Morphological Influence on Chain Dynamics of Polyethylene

The influence of the morphology on the local motion in the noncrystalline regions and chain diffusion between crystalline and noncrystalline regions is studied in ultrahigh molecular weight linear polyethylene: The behaviors of samples of the same material crystallized from the melt and from solution are compared. The geometrical restrictions of the conformational transitions are probed via anisotropic NMR interactions, i.e., 13C−1H dipole–dipole couplings and 13C chemical shift anisotropy. As they are averaged out under MAS, recoupling techniques are applied to yield sideband patterns or quasi-static NMR spectra, from which residual anisotropies are determined. Chain diffusion is probed by 13C exchange NMR. As expected, the local conformational transitions are more restricted in the solution crystallized sample compared with the melt crystallized one. Moreover, the motional narrowing observed by both techniques indicates that the local mobility in the noncrystalline regions of the solution crystallized s...

Probing Structure in Invisible Protein States with Anisotropic NMR Chemical Shifts

A general method for obtaining quantitative structural information on invisible, excited protein states by solution-based NMR spectroscopy is presented. The approach exploits relaxation dispersion techniques in which changes in chemical shifts between ground and excited states are monitored in solutions with and without small amounts of residual molecular alignment. This allows the calculation of differences in chemical shifts induced by alignment that can be directly related to molecular structure, in cases where the orientation and magnitude of the chemical-shift tensor are well defined. An example using carbonyl chemical shifts as probes of a protein-ligand binding reaction is presented to illustrate and validate the method.

Deuterated polymer gels for measuring anisotropic NMR parameters with strongly reduced artefacts

Perdeuterated poly(styrene) is introduced as an almost artefact-free and arbitrarily scalable alignment medium for measuring residual dipolar couplings and other anisotropic NMR parameters; the spectral quality achievable in this new medium is demonstrated for HSQC spectra leading to the conformational analysis of staurosporine and homonuclear TOCSY-type experiments.

Determination of absolute configurations by X‐ray crystallography and 1H NMR anisotropy

To determine the absolute configurations of chiral compounds, many spectroscopic and diffraction methods have been developed. Among them, X-ray crystallographic Bijvoet method, CD exciton chirality method, and the combination of vibrational circular dichroism and quantum mechanical calculations are of nonempirical nature. On the other hand, X-ray crystallography using a chiral internal reference, and 1H NMR spectroscopy using chiral anisotropy reagents are relative and/or empirical methods. In addition to absolute configurational determinations, preparations of enantiopure compounds are strongly desired. As chiral reagents useful for both the preparation of enantiopure compounds by HPLC separation and the simultaneous determination of their absolute configurations, we have developed camphorsultam dichlorophthalic acid (CSDP acid) for X-ray crystallography and 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid) for 1H NMR spectroscopy. In this review, the principles and applications of these X-ray and NMR methods are explained using mostly our own data.

Determination of NMR interaction parameters from double rotation NMR

It is shown that the anisotropic NMR parameters for half-integer quadrupolar nuclei can be determined using double rotation (DOR) NMR at a single magnetic field with comparable accuracy to multi-field static and MAS experiments. The 17O nuclei in isotopically enriched l-alanine and OPPh 3 are used as illustrations. The anisotropic NMR parameters are obtained from spectral simulation of the DOR spinning sideband intensities using a computer program written with the GAMMA spin-simulation libraries. Contributions due to the quadrupolar interaction, chemical shift anisotropy, dipolar coupling and J coupling are included in the simulations. In l-alanine the oxygen chemical shift span is 455 ± 20 ppm and 350 ± 20 ppm for the O1 and O2 sites, respectively, and the Euler angles are determined to an accuracy of ±5–10°. For cases where effects due to heteronuclear J and dipolar coupling are observed, it is possible to determine the angle between the internuclear vector and the principal axis of the electric field gradient (EFG). Thus, the orientation of the major components of both the EFG and chemical shift tensors (i.e., V 33 and δ 33) in the molecular frame may be obtained from the relative intensity of the split DOR peaks. For OPPh 3 the principal axis of the 17O EFG is found to be close to the O–P bond, and the 17O– 31P one-bond J coupling ( 1 J OP = 161 ± 2 Hz) is determined to a much higher accuracy than previously.

NMR in chiral polypeptide liquid crystals: the problem of amines

It is shown that lyotropic liquid crystal mixtures made of poly-γ-benzyl- l-glutamate (PBLG) dissolved in N, N-dimethylformamide (DMF) are efficient anisotropic NMR solvents to distinguish the enantiomers of chiral amines through the effects of the differential ordering of enantiomers. This type of solvent overcomes problems often encountered when dissolving amines into the more conventional PBLG/CHCl 3 or PBLG/CH 2Cl 2 liquid crystals. Furthermore, it is shown that perdeuterobenzyl chloride is an excellent achiral deuterated derivatizing agent for enantiomeric excess measurements of chiral amines in conjunction with the PBLG/DMF solvent.

51V NMR Chemical Shifts Calculated from QM/MM Models of Vanadium Chloroperoxidase

(51)V NMR chemical shifts calculated from QM/MM-optimized (QM=quantum mechanical; MM=molecular mechanical) models of vanadium-dependent chloroperoxidase (VCPO) are presented. An extensive number of protonation states for the vanadium cofactor (active site of the protein) and a number of probable positional isomers for each of the protonation states are considered. The size of the QM region is increased incrementally to observe the convergence behavior of the (51)V NMR chemical shifts. A total of 40 models are assessed by comparison to experimental solid-state (51)V NMR results recently reported in the literature. Isotropic chemical shifts are found to be a poor indicator of the protonation state; however, anisotropic chemical shifts and the nuclear quadrupole tensors appear to be sensitive to changes in the proton environment of the vanadium nuclei. This detailed investigation of the (51)V NMR chemical shifts computed from QM/MM models provides further evidence that the ground state is either a triply protonated (one axial water and one equatorial hydroxyl group) or a doubly protonated vanadate moiety in VCPO. Particular attention is given to the electrostatic and geometric effects of the protein environment. This is the first study to compute anisotropic NMR chemical shifts from QM/MM models of an active metalloprotein for direct comparison with solid-state MAS NMR data. This theoretical approach enhances the potential use of experimental solid-state NMR spectroscopy for the structural determination of metalloproteins.

Stretched Poly(acrylonitrile) as a Scalable Alignment Medium for DMSO

Stretched gels made of cross-linked poly(acrylonitrile) swollen in DMSO are introduced as freely scalable and widely applicable alignment media for the measurement of anisotropic NMR parameters such as residual dipolar couplings. The production process using accelerated electrons for cross-linking is described, NMR signals originating from the alignment medium are characterized, as well as methods for their suppression, and RDC measurements are demonstrated for a number of example molecules.

( R)-(+)-[VCD(+)945]-4-Ethyl-4-methyloctane, the simplest chiral saturated hydrocarbon with a quaternary stereogenic center

Enantiopure ( R)-(+)-[VCD(+)945]-4-ethyl-4-methyloctane, the simplest chiral saturated hydrocarbon with a quaternary stereogenic center, was synthesized by the use of MαNP acid method, and its absolute configuration was first unambiguously determined by the 1H NMR anisotropy, X-ray crystallography, and VCD methods.

Crystalline‐State Conformational Analysis of MαNP Esters, Powerful Resolution and Chiral 1H NMR Anisotropy Tools

AbstractTo compare the crystalline‐state conformations of MαNP acid esters with those existing in the solution state, X‐ray crystallographic analyses of 22 MαNP esters and two MαNP acids were carried out. It was found that all 27 conformers observed in the solid state have structures in which the 2‐CH3 group and the 2′‐H of the naphthyl group are synperiplanar and are almost always located in the same plane. In addition, the CH3–O7–C2–C1 moiety in all cases has an antiperiplanar structure within the MαNP plane. From further analyses, 22 conformers adopt the so‐called syn structure, in which the O7–C2–C1–O6 moiety is synperiplanar. On the other hand, the remaining five conformers have the so‐called anti structure. With regard to the rotational conformation around C1″–O5, all conformers have structures similar to the synperiplanar conformation. In the so‐called syn conformers, the interatomic distance d(H8′–O6) is distributed between 2.48 Å and 2.96 Å, while the distance d(H8′–O7) varies between 2.26 Å and 2.56 Å, indicating the weak bifurcated hydrogen bond between O6–H8′–O7 as the intramolecular stabilizing force. The crystalline‐state conformations of MαNP esters are thus similar to those in solution as determined by 1H NMR spectroscopy, implying that the intramolecular forces make a dominant contribution to the conformations of MαNP esters. The X‐ray crystallographic analyses of MαNP acids indicated that the O6–H(acid)–O7 bifurcated hydrogen bond also serves as an intermolecular force to stabilize the so‐called syn conformation. A similar bifurcated hydrogen bond [O6–H(alcohol)–O7] was observed in a MαNP ester containing a tertiary alcohol group. It should be emphasized that despite the existence of the so‐called syn and anti conformations in the crystalline state, the absolute configurations of all MαNP esters determined here by X‐ray crystallography all agree with those obtained by the 1H NMR anisotropy method.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)